1. Field of the Invention
The present invention relates to a method of detecting the presence of a free polypeptide subunit in the presence of protein which has the subunit as part of its quaternary structure.
2. Brief Description of the Background Art
The structure of proteins can be characterized at several levels of complexity. The primary structure of a protein refers to the sequence of amino acid residues which compose the covalent backbone of the polypeptide chain. The secondary structure of a polypeptide chain is its helically coiled conformation. The tertiary structure of a protein denotes the folded and highly compact conformation of the polypeptide chain. The quaternary structure of a protein results from the aggregation of separately synthesized polypeptide subunits.
Proteins which are composed of more than one polypeptide subunit are known as oligomeric proteins. Examples of oligomeric proteins are the glycoprotein hormones such as, for example, human chorionic gonadotropin; oxygen transport proteins such as hemoglobin; and enzymes such as lactate dehydrogenase.
The glycoprotein hormones in man which are of major medical significance include chorionic gonadotropin (hCG), luteinizing hormone (hLH), folliclestimulating hormone (hFSH), and thyroid-stimulating hormone (hTSH), hCG is synthesized by the placenta during pregnancy whereas the other hormones are synthesized by the anterior pituitary gland. All four of these hormones are structurally related. These hormones are dimeric, composed of two non-covalently associated subunits, denoted alpha and beta. The amino acid composition of the alpha subunit is common to all four hormones, while the beta-subunits, which confer biological specificity, are structurally unique. All of these beta-subunits show some degree of amino acid sequence homology. In man, beta-hCG and beta-hLH are the most closely related, with an amino acid sequence homology of 82%. The amino acid sequence homologies of the beta-subunits of the other glycoprotein hormones range from 25-40%. Of interest in comparing the structure of beta-hCG to beta-hLH is the presence in beta-hCG of a 24 amino acid carboxyterminal polypeptide extension (CTP) which is not present in beta-hLH.
The molecular heterogeneity of hCG and the presence of hCG-related glycoproteins in both normal and malignant tissues has caused major difficulties in studying the regulation of hCG synthesis during pregnancy and the presumed mechanism of hCG genomic derepression in normal and malignant non-trophoblastic cells. The measurement of biologically active hCG levels during pregnancy and the detection of its tumor specific altered forms as biochemical markers of tumor development require the production of highly specific antibodies for either the intact hormone, its subunits, or related forms. Characteristically, most polyvalent antisera directed against hCG cross-react with the other glycoprotein hormones and may not immunologically distinguish hCG from its subunits or related forms. Although the use of highly purified beta-hCG (Vaitukaitis et al., American Journal of Obstetrics and Gynecology, 113: 751 (1972)), chemical analogues of beta-hCG (Pandian et al., Endocrinology, 107: 1564 (1980)), or carboxy-terminal peptides (Matsuura et al., Endocrinoloqy, 104: 396 (1979); Birken et al., Endocrinology, 110: 1555 (1982)) as immunogens may permit the generation of antisera that selectively detect hCG in the presence of hLH, such antisera have thus far been unable to distinguish free beta-hCG subunit from intact native hCG (Armstrong et al., Journal of Clinical Endocrinology and Metabolism, 59: 867 (1984)). Thus, the use of polyclonal antibodies in immunodiagnostic test systems has limitations for proteins such as hCG where there is a group or family of similar proteins with varying degrees of structural homology.
In recent years, considerable research has focused on the use of developing monoclonal antibodies via hybridoma fusion techniques in order to attain the desired specificity. For example, Khazaeli et al. (Endocrinology, 109: 1290 (1981)) reported the production of a monoclonal antibody specific for the beta-hCG subunit which showed conflicting degrees of cross-reactivity with hCG. When cross-reactivity was measured in an ELISA assay in which hCG was immobilized, the authors found a minimal cross-reactivity with hCG of about 2%. When cross-reactivity was measured using a double antibody radioimmunoassay in which radioiodinated beta-hCG competed with increasing concentrations of hCG, the authors state that the monoclonal antibody showed a 0.23% cross-reactivity with hCG.
In Stuart et al., Journal of Endocrinology, 98: 323 (1983), the authors studied the production of monoclonal antibodies to hCG and its subunits. Of the monoclonal antibodies characterized by the authors, one reacted only with intact hCG, and another recognized only the free beta-subunit of hCG.
In Wang et al., Hybridoma, 1: 293 (1982), the authors describe the production of two different hybridomas secreting monoclonal antibodies specific for beta-hCG and alpha-hCG.
David et al. (U.S. Pat. No. 4,376,110) discloses a two-site immunometric assay using monoclonal antibodies from two different clones such that the monoclonal antibodies derived from these clones have specificity for different epitopes of the antigen.
In Givner (U.S. Pat. No. 4,138,214), a method and device for detecting pregnancy is disclosed. The device concentrates a body fluid and detection occurs using a monoclonal antibody specific for the betasubunit of hCG.
None of these references discloses a method for detecting nanogram quantities of free beta-hCG subunit in the presence of microgram quantities of hCG, as is often encountered in clinical specimens.